Dramatic differences have been identified in the ability of breast cancer cells to respond to retinoic acid (RA) both in their ability to continue proliferation and to specifically induce transcription mediated by a retinoic acid response element (RARE). Given the multitude of RA receptors (RAR), isoforms, and other interacting proteins in the RA pathway, this proposal seeks to identify the mechanism of aberrant response using a functional rather than purely genetic or DNA sequence based approach. Initially, efforts will concentrate on four cell lines, two RA high responders and two low responders. 1) Stable transfection of an RARE-chloramphenicol acetyl transferase (CAT) gene into each of these cell lines will provide targets for RA induction assays without the variability associated with transient transfections. A functional RAR will also be stably introduced into these cell lines to explore whether non-responders can be converted to responding cells. 2.) In responsive and nonresponsive cells, RAR transcripts will be examined by hybridization. Expression of receptor proteins will be assesed by immunoblots using recently developed antisera. RA binding to cytosolic and nuclear extracts will be measured by radiolabeled ligand binding studies. 3.) The binding of human RARalpha to specific DNA elements will be measured by gel-shift assays and the effect of adding nuclear and cytosolic extracts from responsive and nonresponsive cells will be investigated. 3) In order to identify genes that are regulated by RA in breast cancer and in normal mammary epithelial cells, differential display polymerase chain reactions will be performed comparing RA responsive, non-responsive, and normal cells. These messages will be cloned and sequenced to determine their identity. Expression of these transcripts will be investigated in primary tissues before and after all-trans RA treatment in a Phase I-II study underway in the Duke Breast Oncology Clinic. In this way, we hope to identify surrogate markers of RA responsiveness which may guide therapy. These studies may identify alterations in gene regulation which are involved in tumor initiation, progression, and which can by used in breast cancer therapy.